The Origin Recognition Complex (ORC), heteromeric six-subunit protein complex, is essential for DNA replication. ORC's functions also extend beyond DNA replication. In our preliminary studies, we found that the Drosophila smallest ORC subunit, Orc6, is important for DNA binding and DNA replication but also has a function in cytokinesis through the interaction with septin protein Pnut. Based on these observations we hypothesize that Orc6 is a multifunctional protein. Orc6 might be involved in a signaling pathway that coordinates many processes of the cell division cycle, such as chromosome duplication and cytokinesis. We hypothesize that Orc6, which bears a structural homology with transcription factor, TFIIB, is essential for targeting of ORC to the origins in Drosophila cells and for the formation of pre- replicative complex. The function of Orc6 in cytokinesis is achieved through the regulation of septin complex activities. To test these hypotheses we plan to analyze at the molecular, cellular and genetic levels the functions and activities of Orc6 and its role in cell cycle. Specifically we will 1) determine the three-dimensional structure of Orc6;2) detertmine functions of Orc6 in DNA binding and DNA replication;and 3) elucidate the role of Orc6 in regulation of septin complex functions. Because ORC is conserved in evolution, what we learn about its functions in Drosophila will be valuable to our understanding of ORC's functions in other eukaryotes. A better understanding of how ORC functions in Drosophila will provide insights into ORC's behavior in mammals and provide clues as to how its misregulation leads to cancer. PUBLIC HEALTH RELEVANCE: Project narrative. The goal of our proposal is to define the role of Origin Recognition Complex (ORC) during the cell cycle in eukaryotes. Because ORC is conserved in evolution, what we learn about its functions will provide insights as to how its misregulation leads to cancer. An understanding of the molecular events involved in the initiation of replication and cell cycle progression will provide a basis for ultimately controlling these processes.